The proposed research focuses on the molecular mechanism of action of the sterol 1,25 dihydroxyvitamin D with major emphasis in understanding the structure and func-tion of the i intracellular receptor protein for this hormone. Despite very low abun-dance of the cellular receptor protein (0.001%), human expression cDNA libraries have been used to clone the full coding resign of the protein. Sequence analysis confirm regions of the gene similar to other cloned receptors thus for (estrogen, progesterone, glucocorticoid, and androgen). The natural gene for the human receptor has recently been isolated and the intron-exon junctions identified. It is proposed to use this information to analyze defective receptor proteins in families with the genetic disease, vitamin D- resistant rickets - type II. Patients with this autosomal recessive disorder display hypocalcemic, hyperparathyroidism and severe osteomalacia/rickets of bone. Cells from these patients will be analyzed for functionality as assessed by hormone binding, nuclear translocation, DNA binding and induction of alternate enzyme (24-hydroxylase) pathways. Genomic libraries will be; constructed from the patient DNA. Screened for receptor containing sequences and sequenced for mutation identification. Restriction fragment length polymorphism (RFlP), polymerase chain reaction (POR) amplification, and ribonuclease A digestion will be employed to identify the patient mutation(s). Subsequently, the identified gene defect (point mutation, deletion, insertion, splice-junction error, promoter defect) will be introduced into the normal clone to recreate the patients mutation. Transfection into COS-l receptor negative cells would be expected to reproduce the identified mutant protein with the same phenotype as isolated from the patient. The in vitro produced defective protein will be tested for functionality (hormone binding, DNA binding, 24- hydroxylase activation) to gain insights into the critically important amino acids of the receptor. Protein DNA contact points will be determined by alkylation interference experiments using dimethylsulfate and ethylnitrosourea. Similarly, mutations involving hormone binding affinity will be studied to define in molecular terms the steroid binding pocket and the critical amino acids involved in ligand interaction on with the receptor.